Background In designing new approaches for the treatment of retinal diseases it is critical to understand their etiology. We have adopted a comprehensive approach, including gene therapy, small molecule screening for drug discovery, and retinal reconstruction. All strategies are guided by the knowledge of molecular mechanisms of photoreceptor development, homeostasis, and disease acquired through other projects (see EY000450-451-473-475). Results 1. Gene therapy In collaboration with P Colosi and T Li we are focusing on the development of gene therapy strategies for the treatment of Leber congenital amaurosis (LCA) due to CEP290 mutations, of autosomal dominant retinitis pigmentosa (ADRP) by NRL knock-down, and retinitis pigmentosa (RP) caused by RPGR mutations. 1.1. LCA We are using 3 vector systems: AAV, lentiviral vectors, and compacted NA strategies. The latter two are being developed in collaboration with M Naash and Oxford Biomedica. A set of AAV CMV mouse CEP290 vectors were generated and co-injected with AAV8 CMV GFP into the subretinal space of Cep290-rd16 mutant mice. Injection of 2e10-2e9 vg/eye produced wide-spread transduction in wild type (WT) retina and rd16 at early time points. However, no therapeutic effect was observed by 8 weeks of age with any of the vectors tested. Work using lentiviral vectors or compacted DNA particles is in progress. 1.2. ADRP We hypothesized that a substantial reduction of NRL expression in adult mouse rods might suppress rod- and induce cone-gene expression. Such a switch could be therapeutic in cases of dominant RP. We generated AAV CMV vectors to express pol 3-driven NRL microRNA upon subretinal injection into 5 months old WT mice. Five weeks post injection rosetting was observed in experimental and control retinas and no change in cone/rod phenotypes. Very high levels of expression of pol 3-driven microRNA constructs might be toxic due to dicer overload when expressed by AAV vectors. 1.3. RP We are focusing on the development of animal models carrying RPGR mutations and displaying rapid degeneration, on the molecular characterization of RPGR gene structure and on the identification of a biologically relevant cDNA for gene therapy to produce clinical human RPGR AAV constructs. 2. Drug discovery We are developing reagents and protocols for high throughput screens of small molecules in collaboration with the NIH Chemical Genomics Center (NCGC) to discover new drugs to prevent photoreceptor degeneration. We are using zebrafish as animal model as well as developing in vitro biomaterial-based tri-dimensional cell cultures. We also plan to use iPS cells to investigate the molecular mechanisms of retinal disease and for small molecule testing. Candidate molecules will be further investigated in preclinical studies using mouse models of retinal degeneration. 2.1. Small molecule screening in zebrafish We have recently strengthened this line of research by hiring a biologist with a background in zebrafish technology. We plan to test small molecules (chemical modifier screen) capable of rescuing photoreceptor degeneration in zebrafish. We have identified two zebrafish mutant lines with photoreceptor degeneration and several GFP/CFP/RFP transgenic lines that would be instrumental in this project. 2.2. Biomaterial-based in vitro cultures We are developing a three-dimensional co-culture system to recapitulate the relationship between photoreceptors and RPE (see below) and that can be adjusted for high throughput small molecule screening. We have thus far successfully addressed the technical challenge of stably incorporating a thin film (collagen vitrigel) into a photopolymerizable hydrogel (poly ethylene glycol PEG). In the next phase, we will encapsulate cells into the PEG matrix, followed by integration of a live RPE cell layer on the collagen vitrigel into the hydrogel matrix. Further efforts will adopt alternative hydrogel systems to allow improved cell motility. We will select cells (photoreceptor and RPE) with degenerative phenotypes relevant to human retinal disease to test libraries of molecules. 2.3. Use of iPS cells to investigate the molecular basis of disease and for small molecule screening. iPS cells from patients or animal models of retinal disease are a valuable source of photoreceptors that can be used to develop assays for small therapeutic molecule screens. Comparative analysis of key characteristics of mutant and WT-iPS lines will be performed to exploit specific differences for the development of high-throughput assays. 3. Retinal Reconstruction 3.1. Biomaterial-based strategies We hypothesize that due to the unique hierarchical, layered structure of the mammalian retina, the position and orientation of the transplanted cells both during and after delivery is critical to optimal cell integration. Furthermore, in view of the distinct polarity that exists between photoreceptors and retinal pigment epithelium (RPE), it is desirable to develop a biomaterials-based in vitro model that approximates the native anatomy and function. Finally, injectable biomaterials may provide structural support to the degenerating retina and therefore aid in vision restoration, particularly in patients with rapid rod degeneration. We are focusing on: assessing biomaterial candidates for their suitability as scaffolds for in vitro cell culture of photoreceptor progenitors and cell transplantation;and evaluating the use of injectable biomaterials for their ability to support the degenerating retina and contribute to vision restoration. We cultured primary retinal cells isolated from developing NRL-GFP mice on biomaterial substrates including collagen vitrigel (gift of T Takezawa) and monitored cell growth, viability, GFP and rhodopsin expression. Preliminary data show more pronounced neuronal morphology and improved cell survival on the biomaterial versus control plates. Other biomaterials (obtained from our collaborators D Kaplan and G Wnek) will be evaluated. We are injecting liquid, photopolymerizable PEG into the subretinal space of developing rd16 mutant mice to test its ability to provide physical support to the degenerating retina. We are currently perfecting the surgical technique, in collaboration with T Li and performing histological evaluations to verify the site of injection. Future experiments will focus on verification of biocompatibility, morphological assessments of retinas post-injection and electroretinography (ERG) to test function. 3.2 Derivation of photoreceptors from Muller glia cell lines We are testing the hypothesis that by introducing NRL in vivo following retinal injury we will enable Muller glia cells to differentiate into rods. Preliminary results obtained by in vivo electroporation of NRL in adult mice were encouraging. More transgenic mice were generated as tools to further test this hypothesis. 3.3 Derivation of photoreceptors from human embryonic stem cell (HESC) lines This project was halted as of August 27, 2010 in compliance with the NIH directive following the Court Case on the use of human embryonic stem cells in Federally funded research. Below is a summary of the accomplishments up to that date. Our goal was to develop optimal conditions for photoreceptor differentiation and identify molecular markers of cells for transplantation.&#8232;NIH-approved HESC lines differentiated to retinal cells and transplanted into the subretinal space of young WT mice differentiated into mature retinal phenotypes by 3 months post-transplantation, as determined by the expression of retinal-specific markers. Differentiation appears to be better accomplished in the subretinal rather than the epiretinal space.